TAS-303 Ameliorates Carbachol-Induced Detrusor Overactivity in Rats, Revealing Its Therapeutic Potential for Overactive Bladder.

Urinary incontinence is defined as an involuntary leakage of urine and is categorized into three types: stress urinary incontinence (SUI), urge urinary incontinence (UUI), and mixed urinary incontinence, which includes symptoms of SUI and UUI. As the underlying mechanisms of SUI and UUI are different, no drug is approved to treat all three types of urinary incontinence. TAS-303 is a selective norepinephrine reuptake inhibitor and has therapeutic potential for patients with SUI. In this report, we describe newly discovered pharmacological properties of TAS-303 and its effects on bladder function. Radioligand binding studies showed that TAS-303 inhibits M3 muscarinic receptor binding, with a Ki value of 547 nM. TAS-303 at 1, 3, and 10 mg/kg dose-dependently prolonged the intercontraction interval of carbachol-induced detrusor overactivity in rats, exhibiting a maximal effect that was comparable to tolterodine. These effects may result from coordinated regulation of bladder afferent activity via M3 muscarinic inhibition and ß3 adrenoreceptor activation by norepinephrine elevation due to norepinephrine transporter inhibition. Moreover, TAS-303 at the effective dose for bladder function did not induce dry mouth or constipation in rats, showing that this compound may have a lower risk of antimuscarinic side effects. Thus, TAS-303 is expected to be a new profile agent with therapeutic potential for all types of urinary incontinence. SIGNIFICANCE STATEMENT: Urinary incontinence is categorized into stress, urge, and mixed urinary incontinence, but because the underlying mechanisms of each differ, no drugs are available that treat all three. TAS-303 has therapeutic potential for stress urinary incontinence. This study describes newly discovered pharmacological properties of TAS-303, which ameliorated bladder afferent activity partly via M3 muscarinic inhibition, indicating improvement in urge urinary incontinence, and highlights the potential of TAS-303 as a new therapeutic agent for all types of urinary incontinence.

Polyelectrolyte Microcapsules Dispersed in Silicone Rubber for in Vivo Sampling in Fish Brains.

Direct detection of fluoxetine and its metabolite norfluoxetine in living fish brains was realized for the first time by using a novel solid-phase microextraction fiber, which was prepared by mixing the polyelectrolyte in the oligomer of silicone rubber and followed by in-mold heat-curing. The polyelectrolyte was finally encased in microcapsules dispersed in the cured silicone rubber. The fiber exhibited excellent interfiber reproducibility (5.4-7.1%, n = 6), intrafiber reproducibility (3.7-4.6%, n = 6), and matrix effect-resistant capacity. Due to the capacity of simultaneously extracting the neutral and the protonated species of the analytes at physiological pH, the fiber exhibited high extraction efficiencies to fluoxetine and norfluoxetine. Besides, the effect of the salinity on the extraction performance and the competitive sorption between the analytes were also evaluated. Based on the small-sized custom-made fiber, the concentrations of fluoxetine and norfluoxetine in the brains of living fish, which were exposed to waterborne fluoxetine at an environmentally relevant concentration, were determined and found 4.4 to 9.2 and 5.0 to 9.2 times those in the dorsal-epaxial muscle. The fiber can be used to detect various protonated bioactive compounds in living animal tissues.

The pH-dependent toxicity of basic pharmaceuticals in the green algae Scenedesmus vacuolatus can be explained with a toxicokinetic ion-trapping model.

Several previous studies revealed that pharmaceuticals with aliphatic amine function exhibit a higher toxicity toward algae than toward other aquatic organisms. Here we investigated the pH-dependent toxicity of the five basic pharmaceuticals fluoxetine, its metabolite norfluoxetine, propranolol, lidocaine, and trimipramine. For all of them, the toxicity increased with increasing pH when aqueous effect concentrations were considered. Since these pharmaceuticals contain a basic amine group that is protonated and thus positively charged at physiological pH and because algae are capable of biological homeostasis, i.e., pH inside the algal cell remains virtually independent of variable external pH, the speciation of aliphatic amines can be different inside the algal cell compared to the external medium. Therefore, we hypothesized that the high toxicity of aliphatic amines in algae is a toxicokinetic effect caused by speciation and not a toxicodynamic effect caused by a specific mode of toxic action. This hypothesis also implies that internal effect concentrations are independent on external pH. On this basis we developed a simple toxicokinetic model, which assumes that only the neutral molecule is bioavailable and can pass the plasma membrane. This assumption is likely to be valid at pH values down to two units below the acidity constant (pK(a)). For lower pH values a more complex model would have to be evoked that includes, an, albeit smaller, permeability of the charged species. For pH>pK(a)-2, we can safely assume that the outer membrane serves as insulator and that the charged species is formed inside the cell according to the pH in the cytoplasm. Thus this toxicokinetic model is an ion-trapping model. The input parameters of this model are the measured aqueous effect concentrations determined as a function of pH and the membrane-water partitioning, which was modelled by the liposome-water partition coefficients of the neutral and cationic species. They were deduced from experimentally determined liposome-water distribution ratios at various pH values measured with an equilibrium dialysis method. The modelled internal effect concentrations were independent of the external pH and effective membrane burdens were in the same range as for other baseline toxicants found in the literature for algae, daphnids and fish. These results confirm that the higher algal toxicity of pharmaceuticals with an aliphatic amine group can be explained by a toxicokinetic effect and that these pharmaceuticals do not exhibit a specific mode of action in algae but act as baseline toxicants.

Determination of fluoxetine and norfluoxetine enantiomers in human plasma by polypyrrole-coated capillary in-tube solid-phase microextraction coupled with liquid chromatography-fluorescence detection.

A sensitive, selective, and reproducible in-tube polypyrrole-coated capillary (PPY) solid-phase microextraction and liquid chromatographic method for fluoxetine and norfluoxetine enantiomers analysis in plasma samples has been developed, validated, and further applied to the analysis of plasma samples from elderly patients undergoing therapy with antidepressants. Important factors in the optimization of in-tube SPME efficiency are discussed, including the sample draw/eject volume, draw/eject cycle number, draw/eject flow-rate, sample pH, and influence of plasma proteins. Separation of the analytes was achieved with a Chiralcel OD-R column and a mobile phase consisting of potassium hexafluorophosphate 7.5mM and sodium phosphate 0.25M solution, pH 3.0, and acetonitrile (75:25, v/v) in the isocratic mode, at a flow rate of 1.0 mL/min. Detection was carried out by fluorescence absorbance at Ex/Em 230/290 nm. The multifunctional porous surface structure of the PPY-coated film provided high precision and accuracy for enantiomers. Compared with other commercial capillaries, PPY-coated capillary showed better extraction efficiency for all the analytes. The quantification limits of the proposed method were 10 ng/mL for R- and S-fluoxetine, and 15 ng/mL for R- and S-norfluoxetine, with a coefficient of variation lower than 13%. The response of the method for enantiomers is linear over a dynamic range, from the limit of quantification to 700 ng/mL, with correlation coefficients higher than 0.9940. The in-tube SPME/LC method can therefore be successfully used to analyze plasma samples from ageing patients undergoing therapy with fluoxetine.

chi-Conotoxin and tricyclic antidepressant interactions at the norepinephrine transporter define a new transporter model.

Monoamine neurotransmitter transporters for norepinephrine (NE), dopamine and serotonin are important targets for antidepressants and analgesics. The conopeptide chi-MrIA is a noncompetitive and highly selective inhibitor of the NE transporter (NET) and is being developed as a novel intrathecal analgesic. We used site-directed mutagenesis to generate a suite of mutated transporters to identify two amino acids (Leu(469) and Glu(382)) that affected the affinity of chi-MrIA to inhibit [(3)H]NE uptake through human NET. Residues that increased the K(d) of a tricyclic antidepressant (nisoxetine) were also identified (Phe(207), Ser(225), His(296), Thr(381), and Asp(473)). Phe(207), Ser(225), His(296), and Thr(381) also affected the rate of NE transport without affecting NE K(m). In a new model of NET constructed from the bLeuT crystal structure, chi-MrIA-interacting residues were located at the mouth of the transporter near residues affecting the binding of small molecule inhibitors.

Noradrenergic mechanisms in cocaine-induced reinstatement of drug seeking in squirrel monkeys.

Norepinephrine (NE) uptake and NE receptor mechanisms play important modulating roles in the discriminative stimulus and stimulant effects of cocaine. The present study investigated the role of NE mechanisms in cocaine priming-induced reinstatement of extinguished drug seeking. Squirrel monkeys (Saimiri sciureus) were trained to stability under a second-order fixed interval, fixed ratio schedule of drug self-administration in which operant responding was maintained jointly by i.v. cocaine injections and presentations of a cocaine-paired stimulus. Drug seeking was then extinguished by replacing cocaine with vehicle and eliminating the cocaine-paired stimulus. In test sessions during which the cocaine-paired stimulus was reintroduced but only vehicle was available for self-administration, priming with cocaine, the dopamine transport inhibitor 1-[2-[bis-(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine (GBR 12909), and the NE transport inhibitors nisoxetine and talsupram induced dose-dependent reinstatement of drug seeking. The maximum effect of the NE transport inhibitors was less than half that of cocaine. Both nisoxetine and talsupram augmented the priming effects of a low but not a high dose of cocaine. The priming effects of nisoxetine were blocked by the alpha1-adrenoceptor antagonist prazosin, the alpha2-adrenoceptor agonist clonidine, and the beta-adrenoceptor antagonist propranolol, but not by the dopamine receptor antagonist flupenthixol. The priming effects of cocaine were antagonized by clonidine and flupenthixol. Neither nisoxetine nor cocaine increased physiological (salivary cortisol) or behavioral (self-directed behaviors) markers of stress. These findings suggest that NE transporter inhibition and alpha2-adrenoceptor mechanisms play a significant role in cocaine-induced reinstatement of drug seeking that is not secondary to activation of brain stress pathways.